This project aims to transform the way in which genes and genomes can be precisely manipulated inside cells by improving the nuclear delivery, efficiency and safety of gene targeting and expanding its applications. Gene targeting is a genetic technique to modify endogenous DNA sequence at will, by changing a mutant DNA sequence into a wild-type copy or vice versa, without removing it from its natural context in the chromosome in a living cell. Gene targeting is therefore a fundamental process not only for functional analysis of genes, proteins and complex biological systems, but potentially also in molecular therapy for the prevention and cure of human genetic diseases originating from specific DNA alterations. The basis of gene targeting is the in situ exchange of genetic information, which in current approaches of genome modification follows the steps of natural homologous recombination. Unfortunately, homologous recombination is active and efficient only in an extremely small set of organisms and cells, where, moreover, it can occur only in a restricted period of the cell cycle. The induction of DNA damage such as double-strand breaks at the targeting locus can partially overcome the low recombination frequencies in many cell types, including human cells. However, the strong threat of unwanted mutations and rearrangements due to both the induction of a break at the targeting region and the occurrence of off-target breaks highly limits applications in gene therapy. In fact double-strand breaks are regarded as one of the primary causes of cancer. The goal of this study is to promote the delivery of novel protein-DNA complexes to the nucleus to promote the reactions of homologous pairing and homologous strand exchange in a non canonical mode without following the steps of homologous recombination, thereby making gene targeting efficient and damage-free in all cells that can be transformed, transfected, or transduced by exogenous DNA. We hypothesize that several proteins among those that normally directly or indirectly interact with chromosomal DNA, if bound to a DNA targeting molecule, can promote delivery and targeting efficiency of this molecule. Our Specific Aims to develop and test this hypothesis are: Aim 1) Test different designs for the DNA targeting molecules and identify the most effective one. Aim 2) Generate DNA-protein complexes containing a site-specific DNA binding protein to drive the DNA template molecule to the targeting locus. We will complex DNA targeting molecules with proteins that are known to bind the DNA in a sequence specific manner in the vicinity of a chosen targeting locus. Aim 3) Identify a set of proteins that, when bound to a DNA template molecule, can [modularly] boost chromosomal gene targeting. This will be achieved via A) screening for gene targeting promoting proteins in yeast cells using a cDNA library generated from yeast cell extract, and via B) screening for gene targeting promoting proteins directly in human cells sensitized to damage repair using a cDNA library generated from cell extract of human cancer stem cells. [C) Testing the identified GTPs for their modular capacity to promote gene targeting.] D) Testing the genomic stability of cells following protein- driven gene targeting. PUBLIC HEALTH RELEVANCE: The best cure/prevention of diseases associated with specific genetic defects as well as the best approach to study gene function can be achieved by precise in situ modification of the desired gene/s, via gene targeting. However, gene targeting is mostly an inefficient process, especially in human cells. DNA double-strand breaks (DSBs) are known to efficiently stimulate homologous targeting. However, cleavage specificity of DSB- inducing enzymes is a major problem since even few off-target DSBs are prone to generate mutations and chromosome rearrangements, which can lead to cancer. Our goal is to develop a modular, cancer-free and efficient gene targeting approach where the DNA targeting molecule is directly driven to its genomic target by a protein that facilitates nuclear delivery and promotes the reaction of strand exchange without inducing DNA damage. This is an exploratory study and the results obtained will provide a basis for a future R01 submission focused on mechanisms of gene targeting guided by proteins and on broader application for gene and genome modification and gene therapy.